HAL Id: hal-00892090
https://hal.archives-ouvertes.fr/hal-00892090
Submitted on 1 Jan 2010
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of
sci-entific research documents, whether they are
pub-lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
America
Thomas E. Rinderer, Jeffrey W. Harris, Gregory J. Hunt, Lilia I. de Guzman
To cite this version:
Review article
Breeding for resistance to Varroa destructor
in North America*
Thomas E. R
inderer
1, Je
ffrey W. H
arris
1, Gregory J. H
unt
2, Lilia I. de G
uzman
11USDA- Agricultural Research Service, Honey Bee Breeding, Genetics and Physiology Laboratory,
1157 Ben Hur Road, Baton Rouge, LA, 70820, USA
2Dept. of Entomology, Purdue University, West Lafayette, IN 47907, USA
Received 24 September 2009 – Revised 1 January 2010 – Accepted 30 January 2010
Abstract – Breeding for resistance to Varroa destructor in North America provides the long-term solution to the economic troubles the mite brings. This review reports the development of two breeding successes that have produced honey bees of commercial quality that do not require pesticide treatment to control
Varroa, highlights other traits that could be combined to increase resistance and examines the potential uses
of marker-assisted selection (MAS) for breeding for Varroa resistance. Breeding work continues with these stocks to enhance their commercial utility. This work requires knowledge of the mechanisms of resistance that can be further developed or improved in selected stocks and studied with molecular techniques as a prelude to MAS.
Varroa resistance/ breeding program / Russian honey bees / Varroa-sensitive hygiene / marker-assisted selection
1. INTRODUCTION
The introduction of Varroa destructor Anderson & Trueman (2000) into North America during the late 1980s caused dra-matic changes to beekeeping practices and in-creased the costs of honey production and pol-lination. Increased costs stemmed primarily from the control measures necessary to pre-vent loss of colonies from varroosis. Most bee-keepers relied on acaricides such as Apistan
r
(fluvalinate) or CheckMiteTM (coumaphos)to control Varroa mites. Unfortunately, use of chemicals has led to the development of acaricide-resistant mites and to increased residues of chemicals in beeswax and honey.
A variety of non-chemical control meth-ods were developed to circumvent or de-lay the problems of acaricide-resistant mites and chemical residues in beekeeping products.
Corresponding author: T. Rinderer, Tom.Rinderer@ars.usda.gov * Manuscript editor: Marla Spivak
Non-chemical controls for Varroa mites in-cluded mite trapping by removal of capped drone brood, screened floors, sticky traps on the bottom board, and use of Varroa-resistant honey bees. Using Varroa-resistant honey bees is ideal since the need for acaricides is either reduced or eliminated without a need for addi-tional Varroa control measures.
Breeding for Varroa-resistant honey bees became the primary goal for a number of re-search groups around the world. Within North America, Varroa resistance has been produced by at least three breeding programs. One program from the University of Minnesota produced measurable Varroa resistance as a consequence of selecting for improved gen-eral hygienic behavior (Boecking and Spivak,
1999; Spivak and Reuter, 2001a, b; Ibrahim et al.,2007). The “Minnesota Hygienic” stock (MNHYG) is sold commercially throughout the US (Spivak et al., 2009). Two other pro-grams were initiated at the USDA-ARS Honey Bee Breeding, Genetics and Physiology Labo-ratory in Baton Rouge, LA, and they are the
primary focus of the current review. The Rus-sian Honey Bee (RHB) Program and the
Var-roa-Sensitive Hygiene (VSH) Program were
initiated specifically to produce Varroa resis-tant honey bees that would be suitable for com-mercial use.
Although they differ in general breeding ap-proach, the two programs have produced and released Varroa-resistant honey bees that are sold commercially. These honey bees require substantially fewer acaricide treatments for controlling Varroa mites, and they retain the commercial qualities desired by beekeepers. Both programs have relied upon traditional breeding techniques and an understanding of the known mechanisms of Varroa resistance. One goal is that future selection will include the use of molecular genetics. Specifically, the development of marker-assisted selection (MAS) will likely accelerate breeding progress in these two programs and programs that are currently developing other Varroa resistance traits such as nestmate grooming.
2. MECHANISMS OF RESISTANCE The Russian (or Korean) haplotype of V.
destructor is the hypervirulent variant which
threatens Apis mellifera beekeeping world-wide (de Guzman et al.,1997,1999; Anderson and Trueman,2000). Honey bee colonies that survive infestations of this Varroa haplotype have one or more behavioral or physiological traits which underlie their resistance to Varroa.
2.1. Behavioral mechanisms of resistance
2.1.1. Hygienic behavior
Hygienic bees are able to detect, uncap and remove diseased brood (Rothenbuhler,
1964; Gilliam et al.,1983; Spivak and Reuter,
2001b). A general test of hygiene, the removal of freeze-killed brood by colonies (Spivak and Reuter,1998), correlates relatively well with removal of Varroa-infested brood (Boecking and Drescher,1992; Spivak, 1996). Removal of mite-infested brood is well established in A.
cerana (Peng et al.,1987).
The ability to remove brood infested with
Varroa has been bred to high levels in A. mellifera colonies bred for VSH (Harbo and
Harris, 2005; Harris, 2008). VSH is more pronounced in infested worker brood than in drone brood suggesting that increased mite in-festation may occur in VSH colonies when drone brood is abundant (Harris, 2008). As VSH bees uncap and remove infested brood, freed adult female mites usually transfer onto the bees removing the brood (Aumeier and Rosenkranz,2001) but may eventually become free on the combs and exposed to attack by bees. Thakur et al. (1997) documented that honey bees can detect, grab and bite free-moving mites. The mites may also become phoretic and exposed to grooming. Hence, VSH may be a basic mechanism which can enhance other traits such as nestmate groom-ing and an increased phoretic period (Ibrahim et al.,2007).
2.1.2. Grooming behavior
Honey bees clean themselves (autogroom-ing) and nestmates (allogroom(autogroom-ing) (Haydak,
1945). Grooming may injure or kill Varroa mites (Ruttner and Hänel, 1992), or it may cause mites to either move to other parts of the autogroomer’s body, transfer to a new host or be removed from the bee’s body without causing visible injury (Büchler et al., 1992). Grooming is rarely observed directly. How-ever, variation among honey bee stocks in grooming has been inferred from the propor-tion of mites that drop to hive floors that are damaged, apparently from bees’ mandibles (Boecking and Spivak,1999; Fries et al.,1996; Rinderer et al., 2001a; Arechavaleta-Velasco and Guzman-Novoa,2001).
and found that the two best predictors of re-duced MPG were the average number of mated female mite offspring and the proportion of mutilated mites (Mondragon et al.,2005). This is one of only two studies in North Amer-ica that have used diverse queen sources and investigated mechanisms of resistance. The other study identified VSH as the primary mechanism (see below).
Grooming is a heritable trait (Moretto et al.,
1993). However, its usefulness in a breeding program is controversial (Rosenkranz et al.,
1997; Bienefeld et al.,1999; Aumeier,2001). Several measurement problems have been identified. Usually, the proportion of damaged mites, a difficult and time consuming measure-ment, is the only criterion considered. How-ever, living apparently uninjured mites have been detected in high numbers on bottom board traps (Fries et al., 1996). They may also indicate grooming and actually be in-jured or debilitated (Thakur et al.,1997). Al-ternately, they may be healthy, fallen owing to hot weather (Webster et al.,2000).
Injuries to mites may result from: (a) grooming, (b) removal of dead mites (Rosenkranz et al., 1997; Bienefeld et al.,
1999), or (c) predation by wax moth larvae and ants (Szabo and Walker, 1995). Davis (2009) asserted that indentation on the mites’ idiosoma is not damage caused by bees but is acquired during mite development. However, pieces missing from the idiosoma and missing legs cannot be attributed to normal mite devel-opment. Laboratory assays of grooming using either individual bees or cages of bees have been developed and produce promising results that correlate with the proportion of damaged mites in source colonies, thus circumventing the difficulty of evaluating damaged mites (Arechavaleta-Velasco and Guzman-Novoa,
2001; Currie and Tahmasbi,2008; A. Gandino and G.J. Hunt, unpubl. data). An efficient laboratory assay should improve measurement precision and accelerate selection.
2.1.3. Removal of mites from the nest
Morse et al. (1991) hypothesized that bees may carry and discard Varroa mites outside
the nest. Lodesani et al. (1996) confirmed this hypothesis using external traps (Gary,1960). Likewise, living Varroa mites can be lost dur-ing foragdur-ing flights (Kralj and Fuchs, 2006) and more frequently by RHB (Kralj, 2004). Kralj also observed that a higher proportion of the infested RHB did not return to the hive as compared to infested A. m. carnica and inter-preted the behavior to be an adaptive contribu-tion to resistance (Kralj and Fuchs,2006).
2.2. Physiological mechanisms of resistance
2.2.1. Brood characteristics
Brood attractiveness – The use of this character in breeding programs appears ques-tionable because several comparative stud-ies exposing different A. mellifera brood yielded contradictory results (Büchler, 1990; de Guzman et al., 1995, 1996; Calis et al.,
2006). Attractiveness is commonly measured as the percentage of cells infested, but mea-surements of the reproductive potential of in-festing Varroa mites may be more useful. Higher rates of non-reproduction (NR) in VSH bees (Harbo and Hoopingarner, 1997) and RHB (de Guzman et al., 2007) by infest-ing mites may result from reduced brood at-tractiveness but may also be a direct result of VSH. However, less attractive hosts may also result in a reduced reproductive success among reproductive mites as found with RHB (de Guzman et al., 2008) and other stocks (Camazine, 1986; Harbo and Hoopingarner,
1997; Ibrahim and Spivak, 2006). This re-duced reproduction might be a useful trait for breeding.
larvae of A. cerana or both worker and drone larvae of A. mellifera (Xing et al.,2007). Cop-per and zinc are important for insect growth and fecundity (McFarlane,1976).
Larval food and comb properties – Traits for selection may also include the chemistry of comb and larval food. Cocoons contain semio-chemicals used for the deposition of Varroa feces (Donze’and Guerin,1994). Larval food may also contain chemicals that attract or in-fluence Varroa mite reproduction (Nazzi et al.,
2001). De Guzman et al. (2008) showed that the comb built by RHB contributed to an in-creased rate of NR, and dein-creased numbers of progeny and viable female offspring.
2.2.2. Phoresy
If mites in some bee colonies are phoretic for longer periods, they may have fewer chances of reproducing during their life and an increased potential for being groomed (Ruttner and Hänel, 1992). Selection for in-creased phoresy would enhance mite resis-tance. However, phoresy may be influenced by other resistance traits. VSH reduces the num-ber of mites in brood, and grooming reduces the number on adults. The mites released by VSH may either die or join the phoretic pop-ulation, but in either case, the proportion of phoretic mites increases. In RHB, phoresy may be influenced by brood unattractiveness (de Guzman et al., 2007), winter- or nectar-dearth induced broodlessness (Tubbs et al.,
2003), or be supplemented by increased and prolonged drone production (Rinderer et al.,
2001a; de Guzman et al.,2007).
3. SELECTIVE BREEDING OF VARROA-SENSITIVE HYGIENE (VSH)
3.1. History of the suppression of mite reproduction (SMR)/VSH breeding program
This breeding program sought to identify and enhance traits of honey bees that limit
growth of Varroa populations from bee stocks that were already in the U.S. The primary goal is to deliver useful Varroa resistance traits to the beekeeping industry by providing highly selected germplasm that can either be intro-gressed by selective breeding into existing commercial stocks or outcrossed to produce hybrid bees that retain significant Varroa re-sistance.
Early in the breeding program (1996– 2001), selection for Varroa resistance fo-cused on MPG among homogeneous infested colonies over about ten weeks (Harbo and Har-ris,1999a). Resistance was defined as the abil-ity of a colony to retard MPG. MPG was estimated by an exponential growth equation (Branco et al.,1999), and environmental vari-ation (Harris et al.,2003) was minimized by forming colonies at the same time and within the same apiary. Success in finding genetic dif-ferences among colonies was enhanced by the use of single drone inseminations of queens. This mating technique produced workers of one patriline with reduced genetic variation within a colony, which allowed variation be-tween colonies of diverse genetic backgrounds to be more apparent (Rothenbuhler, 1960). Of the four mechanisms of resistance [post-capping period, freeze- killed brood removal, grooming and NR] that were measured, only NR was found correlated with MPG (Harbo and Hoopingarner, 1997; Harbo and Harris,
1999a).
NR was caused by two heritable traits (Harbo and Harris,1999b). Brood from queens with resistance genes caused increased NR, but the strongest effect came only after adult worker bees had been produced from the queens (Harris and Harbo, 2000). Breeding for the adult bee effect was favored because it had the strongest influence on Varroa resis-tance (Harbo and Harris, 1999b). This adult bee effect was called the SMR trait (Harbo and Harris,2002). Beginning in 2001, SMR lines were selected for increased NR (Harris and Harbo,1999). The brood effect still occurs in
3.2. Mechanism of resistance in VSH bees
Selection for high percentages of NR mites continued until 2005 when it was discovered that infertility of mites was linked to hygienic removal of mite-infested pupae (Harbo and Harris,2005; Ibrahim and Spivak,2006). Be-cause VSH is the primary mechanism of resis-tance, the name replaced SMR (Harris,2007). The new understanding came when Ibrahim and Spivak (2006) observed that colonies of VSH bees removed freeze-killed more quickly than the MNHYG stock of bees, and so were more hygienic. Also, it was found that the in-fertility of mites in foreign brood increased af-ter a 1-week exposure to VSH bees (Harbo and Harris, 2005). Increased mite infertility was correlated with a decrease in the brood infes-tation rate, which presumably resulted from VSH. This could be explained if VSH bees preferentially removed pupae that were in-fested by mites with offspring rather than pu-pae with infertile mites (Harbo and Harris,
2005,2009).
However, recent experiments indicated that VSH bees remove mite-infested pupae whether mite offspring are present or not (Harris et al., 2009, 2010). Therefore, in-creased NR is likely caused by other as-pects of hygiene. For example, uncapped pu-pae are sometimes recapped by non-hygienic bees within a hygienic colony (Arathi et al.,
2006), and this frequently occurs in VSH colonies (Harris,2008). Perhaps reproduction by Varroa is disrupted by the uncapping of the brood cells, and some uncapped pupae are recapped with NR mites inside them. Be-cause VSH bees remove mite-infested pupae without regard to the presence of mite off-spring, it seems unlikely that the stimulus trig-gering VSH is related to oviposition or to odors from mite offspring. Neither odors nor movements of adult Varroa mites elicit re-moval of mite-infested brood (Aumeier and Rosenkranz,2001). Therefore, the stimulus for VSH probably originates from odors of in-fested hosts (Martin et al., 2002); however, there are other possible triggers for the re-moval of mite-infested brood, and the specific cues remain unknown (Vandame et al.,2002).
A key goal has been to develop more ef-ficient methods for breeding the VSH trait. Selection for SMR involved field tests last-ing 2–6 months. New understandlast-ing of VSH as a behavior of adult bees may allow ac-celerated selection based on direct measure-ments of behavior. However, current selection focuses on indirect measures of behavior such as a reduced Varroa infestation of brood after exposure to bees. The quickest bioassays for VSH involve the introduction of infested for-eign brood into VSH colonies for either 40 h or one week (Harris,2007; Villa et al.,2009a). The 40-h exposure showed a strong correla-tion between reduccorrela-tion in brood infestacorrela-tion and MPG, while strong correlations between reduced brood infestation, mite fertility, and MPG were apparent after the 1-week expo-sure. Currently, a 1-week exposure of brood to colonies is the primary method recommended for assessing VSH in breeding stock.
3.3. Performance of VSH bees in commercial beekeeping environments
The strongest VSH expression comes from purely mated queens, but early in the program some colonies of a VSH× VSH mating devel-oped a poor brood pattern. The poor brood pat-terns were not related to a sex allele problem from inbreeding (Harbo and Harris,2001). We know this because brood viabilities were often very high (>85%) for queens when they first begin egg-laying, and it is only after several months that poor brood patterns developed. The cause of poor brood production is not un-derstood, but not all VSH lines developed the problem (Harbo,2001). The problem is not in-herent to queens or brood, and it can be selec-tively bred out of VSH lines while retaining
Varroa resistance (Tom Glenn, unpubl. data).
For example, pure naturally mated and hybrid VSH queens did not develop poor brood pro-duction over a 3-year field trial (Ward et al.,
2008). Until there is a better understanding of the problem, commercial release of VSH through hybrids is recommended.
brood production and colony size during rou-tine maintenance of experimental lines. Hy-brid VSH bees grew half of the mite pop-ulations of control colonies, and their adult bee populations and brood areas were larger (Harbo and Harris, 2001). Mite populations in hybrid VSH colonies were slower to reach an economic threshold in a 1-year study, but some of them developed poor brood produc-tion (Delaplane et al.,2005). Over a 2-year pe-riod the Varroa resistance of MNHYG was sig-nificantly increased in hybrids having less than the typical F2contribution from VSH parents
(Ibrahim et al.,2007).
The performance of either hybrid VSH or pure VSH colonies was compared to RHB and a commercial control stock in beekeep-ing operations in Alabama over a 3-year period (Ward et al.,2008; Danka et al.,2008). Over the entire study, only 12% of VSH colonies reached a recommended treatment threshold (Delaplane and Hood,1999), whereas 24% of RHB and 40% of the controls exceeded thresh-old, although treatment thresholds for resis-tant bees are not established and may be dif-ferent. The stocks were similar in colony size, honey production and queen survival (Ward et al., 2008). Strong Varroa resistance can be obtained by using VSH honey bees with-out any significant loss of desired beekeeping characteristics. Beekeepers reported good bee-keeping quality for all stocks, even pure VSH colonies.
3.4. Transfer of VSH germplasm to the beekeeping industry
High-VSH germplasm currently is released to the beekeeping industry through Glenn Apiaries (www.glenn-apiaries.com). Selected breeder queens containing the VSH trait are distributed to queen producers who raise daughters from the breeder queens and out-cross them to unselected drones. In this way, significant Varroa resistance is delivered in the form of hybrid VSH colonies, while brood production and other desired beekeeping qual-ities are retained. VSH breeder queens have been sold to 50–80 queen producers in the US during each of the last few years, and about
12–15 of these queen producers sell a variety of outcrossed VSH queens to beekeepers (T. Glenn, unpubl. data).
Selection and breeding of VSH bees have been focused mainly on Varroa resistance, with some selection to avoid susceptibility to tracheal mites. The research lines maintained by us have been variable for other charac-teristics. Further breeding would be desirable in several areas. For example, recent surveys of beekeepers indicate a strong preference for Italian honey bees (T. Glenn, unpubl. data). Initial efforts have been made in the VSH breeding program to select for characteris-tics associated with Italian stock. Additionally, some VSH lines will be selected for improved performance in migratory pollination service.
Although high expression of the VSH trait can control growth of mite populations, re-liance on a single resistance mechanism may be unwise. A goal of additive Varroa resistance produced by combining multiple mechanisms is highly desirable. Several of the other mech-anisms of Varroa resistance are currently be-ing selected by us. We are also trybe-ing to iden-tify the semiochemicals that elicit removal of mite-infested brood and molecular markers as-sociated with the VHS trait that could be used in future selective breeding. Until fully Varroa resistant bees have been developed, the VSH-trait should be part of a comprehensive inte-grated pest management scheme (Delaplane et al.,2005).
4. THE USDA-ARS RUSSIAN HONEY BEE (RHB) BREEDING PROGRAM 4.1. Early evaluations in Russia
and the United States
The RHB breeding program has developed a novel stock, derived from the honey bees of far-eastern Russia, which is resistant to V.
de-structor. These honey bees were brought there
from Western Russia in the mid-1800s by pi-oneers (Crane, 1978). The area is within the home range of A. cerana, the original host of V.
quickly, producing the historically longest as-sociation of A. mellifera and Varroa. It was hy-pothesized that this long association gave the best chance for natural selection to mold honey bees resistant to Varroa (Danka et al.,1995). Exploring this hypothesis led to the develop-ment of the RHB stock.
Collaborative research (Danka et al.,1995) with the Far-Eastern Branch of the Russian Academy of Sciences resulted in surveys and a natural history comparison of Varroa MPG in Russia and the US which suggested that RHBs perhaps were comparatively resistant to
Varroa. Consequently, honey bee stock from
Russia was imported through quarantine into the US. Confinement on the island quarantine lasted eight months where the imported RHB were subjected to rigorous regulatory inspec-tion (Harris et al.,2002).
After quarantine, the RHB colonies were uniformly inoculated with Varroa and eval-uated for MPG. Most colonies supported a MPG lower than expected for susceptible colonies. Many had a MPG that was half to a 10th of the standard, with one colony not showing any MPG. Forty of the queens were chosen to be further evaluated in a sib-test (Rinderer et al.,1999).
Although the tests of individual queens pro-vided additional evidence that the RHBs were resistant to Varroa, a rigorous experiment to compare the RHBs with known susceptible honey bees in a side-by-side experiment was lacking. Consequently, a comparative exper-iment was begun (Rinderer et al., 2001a). Newly produced RHB and Italian queens se-lected for resistance to Varroa were estab-lished in colonies inoculated with Varroa mites. The colonies were evaluated for num-bers of adult female Varroa and the presence of varroosis from June, 1998 to November, 1999. The average numbers of adult female Varroa in Italian colonies continually grew to about 10 000 in the summer of 1999 (Fig. 1). The average number of mites in RHB colonies also grew, but only to about 4000 during this time. By July of 1999, all of the Italian colonies had died, most of them exhibiting varroosis, and all of them having high numbers of mites while only three RHB colonies died, apparently be-cause of varroosis. The comparative survival
n
Figure 1. Average Varroa infestations (estimates of the total number of adult female mites) in RHB (white bars) and Italian colonies (black bars) through time. Error bars = sem (From: Rinderer et al.,2001a).
of the RHB colonies, the comparatively fewer mites infesting the RHB colonies and the de-cline in mite numbers in late-summer and au-tumn all supported the conclusion that RHBs were resistant to Varroa.
4.2. Mechanisms of RHB resistance to mites
Comparative studies of RHB and Italian honey bees found several mechanisms under-lying RHB’s resistance to Varroa mites. RHB consistently had low proportions of brood in-fested (Rinderer et al., 2001b; de Guzman et al., 2007; de Guzman et al., 2008) and fewer multiply infested cells in both worker and drone brood (de Guzman et al., 2007). A reduced attractiveness of RHB brood and a strong expression of hygiene (de Guzman et al., 2002) may have contributed to the in-creased rate of non-reproductive mites and de-creased number of progeny and number of viable female offspring in RHB (de Guzman et al., 2008). Decreased reproductive suc-cess also may have been increased by the combs built by RHB (de Guzman et al.,2008). Reduced brood attractiveness and a higher rate of brood removal may have contributed to the extended phoretic period of Varroa mites in colonies of RHB (Rinderer et al.,
vulnerability of Varroa mites to be groomed in RHB colonies. RHB colonies had a higher proportion of damaged mites (42% vs. 28%) on bottom board traps than did Italian bees, which suggests that they have a strong Varroa grooming trait (Rinderer et al.,2001a).
In addition to having resistance to
Var-roa, RHBs were found to have other valuable
traits which have been maintained or improved through selection. First, they are highly re-sistant to Acarapis woodi (de Guzman et al.,
2001). Resistance to A. woodi was a contribut-ing factor to comparatively very high winter survival of RHBs (de Guzman et al., 2005; Villa et al., 2009b). Resistance to A. woodi in RHBs is attributed to autogrooming (Villa,
2006), which might also contribute to resis-tance to Varroa. The genetic control of auto-grooming is polygenic with some of the genes having a strong dominance effect (Villa and Rinderer,2008). Also, RHBs are very hygienic (de Guzman et al.,2002) according to a stan-dardized test (Spivak and Reuter,1998).
4.3. Selection procedures
Testing and stock selection began with co-operating beekeepers who provided apiaries in northeastern IA, known for having both harsh winters and perennial problems with tracheal mites, apiaries in central MS that experienced a mid-summer soybean (Glycine max) nec-tar and pollen flow and apiaries in southern LA that experienced a late spring Chinese tal-low (Triadica sebifera) nectar and pollen ftal-low. These test apiaries provided a diversity of con-ditions that allowed the selection program to produce a stock adapted to a wide range of beekeeping. On occasion, a line would prove exceptional in one area but poor in a different area and was discarded.
Inclusion of stock into a closed breeding population based on selection began in 1999 and continued to 2007. Daughters of the best of the queens imported from Russia were sub-jected to an intensive sib-test in the nine api-aries supplied by the cooperating beekeepers. Overall, daughters of 42 queens identified by individual tests of the 362 queens imported from Russia were evaluated in sib-tests, and
18 queen lines (5%) were included in the closed breeding population. Sib-tests evalu-ated MPG in the colonies and their honey production. Data for each colony were con-verted to within apiary Z-scores, permitting the comparison of lines and colonies among all apiaries. Using these comparisons, some lines were chosen for potential inclusion into the closed population of breeder lines using an un-weighted selection index score which com-bined each colony’s Z-score times –1 for MPG and the Z-score for honey production to pro-duce a single number for comparisons between colonies (Rinderer et al., 2001b). These lines were further tested to assure A. woodi resis-tance using a standardized test (Gary and Page,
1987). Lines not highly resistant to A. woodi were culled regardless of their selection index score.
Selection for mite resistance was based solely on colonies having low MPG. Any mechanism that promotes reduced MPG would be selected for using this criterion. Some mechanisms of resistance to Varroa might be associated with colonies being too small to be commercially useful (Büchler,
1997). However, lines were also concurrently selected for increased honey production which acts as a reasonable counter measure to pre-vent colonies from simply getting smaller as a response to selection for reduced MPG. Both selection criteria are broad. Any specific trait that contributes to a reduction in MPG would potentially be enhanced by selection. Like-wise, any trait that generally enhances fitness would potentially be enhanced by selection for honey production.
4.4. Development of a closed breeding population of RHB
mated to drones of the other two groups of lines. This plan is designed to reduce inbreed-ing while also providinbreed-ing a practical method to arrange the open mating of 18 separate lines on an isolated island. Queens of one group can be produced and mated simultaneously. This mating scheme has resulted in a stock that re-tains good genetic diversity among groups and lines (Bourgeois et al.,2008). Also, allelic fre-quency differences at molecular loci enable RHBs to be distinguished from other commer-cial stocks with very high accuracy (Bourgeois and Rinderer,2009). Using this suite of loci, the diversity among RHBs compares favorably to the diversity of non-RHB stocks in the US.
Between 1999 and 2007, the program em-phasized sib-testing of lines that could po-tentially be added to the closed population. However, each year those lines that had been added to the program were tested and prop-agated. Owing to limited resources only be-tween 8 and 12 colonies were used in tests of each line. However, in 2001 one line was included in the trials for the next three years as a test of the success of selection. The line had a comparatively high average Z-score for honey production and a moderately negative z-score for MPG (negative being desirable). When the scores were combined in an un-weighted selection index, the line ranked high-est of the lines thigh-ested that year. Each year the MPGs for the line were lower than the previous year (Fig. 2) suggesting that selec-tion improved resistance to Varroa. The line continued to be the best honey producer in subsequent years, but honey production was not substantially improved. A separate ex-periment (de Guzman et al.,2007) compared RHB colonies from lines in the closed popu-lation to Italian colonies from 2001 to 2003 in the same apiary. Each year new queens were used. MPGs for Italian colonies were al-ways larger and varied among the years with-out having a year to year trend. MPGs for RHB colonies trended lower through the years (Fig. 3). Hence, selection within the closed population increased the stock’s resistance to
Varroa.
Improvements in honey production are less well documented. However, honey production by RHBs has equaled or surpassed the honey
Figure 2. Changes in Z-scores of mite popula-tion growth estimated from a sib-test in four years for Italian colonies, a RHB line (selected Russian) that underwent selection for reduced mite popu-lation growth and a group of RHB lines (unse-lected Russian) being evaluated for inclusion into the closed breeding population. The selected line became comparatively more resistant each year.
Figure 3. Instantaneous mite population growths (IMPG) across three years for an Italian stock not selected for resistance to Varroa and lines of RHBs in a closed breeding population that were se-lected for reduced Varroa population growth (from data presented in: de Guzman et al.,2007). IMPG showed annual decreases for the selected RHB but not for the unselected Italian stock.
production of a well respected Italian stock of honey bees in several experiments (Rinderer et al., 2001c, 2004). These results contrast with European studies that found “Primorski” honey bees were resistant to Varroa but pro-duced less honey than locally selected A. m.
be less gentle, although these studies included most lines of RHB and their hybrids rather than only lines released for general distri-bution. Some RHB hybrids are not gentle. However, purebred lines released for general distribution overall have acceptable traits in-cluding honey production and gentleness.
4.5. Transfer of RHB to the beekeeping industry
The breeding and selection program con-tinues as a commercial activity. A group of United States queen breeders have formed the Russian Honeybee Breeder’s Association and are continuing the selective breeding of the stock (Brachman,2009). Members of this group and their customers use no other method to control Varroa beyond using the stock and have done so for many years. This manage-ment is consistent with studies of the stocks resistance (Rinderer et al., 2003,2004). Ad-ditionally, RHB’s outcrossed to susceptible stock express enough resistance to permit re-duced schedules of Varroa control treatments (Harris and Rinderer,2004).
5. MOLECULAR GENETIC
APPROACHES TO RESISTANCE BREEDING
The publication of the sequence of the honey bee genome provided a means to study all of the genes in the bee (Honey Bee Genome Sequencing Consortium,2006). How can molecular genetics help us to breed bees that resist mites? If we could identify the genes that influence resistance, we could se-lect alleles directly by looking at the bee’s DNA (marker-assisted selection: MAS). One approach is to use microarrays to study gene expression in resistant and susceptible lines to identify resistance genes, and one study us-ing Varroa-survivus-ing bees in France has been conducted (Navajas et al.,2008). Microarrays have been very useful for characterizing gene expression patterns for behavioral and phys-iological states, suggesting genes that may influence them (e.g., Whitfield et al., 2003;
Grozinger et al.,2007). But microarray studies would not necessarily identify the genes that need to be selected for resistance. It is quite possible that the gene(s) responsible for resis-tance is not among them since it may control the expression of other genes, or is only differ-entially expressed at certain times, or in a spe-cific tissue. Another difficulty for microarrays comes from effects of genetic backgrounds on gene expression, since inbred lines are diffi-cult to develop for honey bees and resistant and susceptible strains would have many ge-netic differences unrelated to resistance. Prob-ably the greatest benefit of differential gene ex-pression studies is to identify genes involved in physiological processes that occur during mite infestation and the development of par-asitic mite syndrome (Navajas et al.,2008).
Another possibility is to identify chromo-somal regions that contain genes influencing a trait. The technique of quantitative trait lo-cus (QTL) mapping is applicable to any heri-table trait. The number of QTLs affecting the trait, their relative effects and their locations on chromosomes can be estimated. Basically, this involves studying the quantitative trait’s association with DNA markers in a family of individuals descended from a hybrid individ-ual derived from a cross between parents hav-ing low and high phenotypes of the trait (e.g. resistant and susceptible). The assumption is that there are multiple genes influencing the trait and that certain DNA markers that are close to genes influencing the trait will be non-randomly associated with the trait values, de-spite crossing over during meiosis (recombi-nation). Recombination is the basis for genetic mapping because the number of crossovers be-tween two points on a chromosome correlates with their physical distance. A honey bee ge-netic map revealed a higher rate of meiotic re-combination than any reported for a higher eu-karyote (Hunt and Page,1995; Solignac et al.,
2004). This high rate of recombination is very useful for QTL mapping because it results in higher resolution of physical chromosome dis-tance which reduces the effort required to find which gene(s) is influencing the trait.
age, response to sucrose and ethanol, worker egg-laying, and even ability to learn (Hunt et al.,1995,1998; Page et al.,2000; Chandra et al.,2001; Arechavaleta-Velasco and Hunt,
2004; Rueppell et al.,2004, 2006; Ammons and Hunt, 2008; Oxley et al., 2008). If the sequences of DNA fragments used as mark-ers are known, it is possible to identify the trait’s candidate genes. For pollen-foraging and stinging, the QTL regions each contained about 40 genes (reviewed by Hunt et al.,
2007). Regarding traits that influence resis-tance to Varroa, one study identified seven pu-tative QTLs influencing general hygiene, but the markers were of unknown sequence so it was not possible to align the genetic map with genome sequence or to identify candidate genes (Lapidge et al.,2002).
What are the future prospects for using MAS? MAS, is most valuable when the cost of determining the phenotype (resistance) is high, and the time between generations is long (Hospital,2009). Traits such as mite-grooming and VSH appear to be the most desirable
Var-roa resistance traits but are difficult to
mea-sure. Also, measurement of trait expression requires full colonies. MAS may permit by-passing the production of colonies and thereby speed selection while insuring the presence of the right alleles of specific genes. On the other hand, because of the high recombination rate of the honey bee we may require markers that are within the actual gene sequence, and the gene would need to be identified. Single-nucleotide polymorphisms (SNPs) often are found within honey bee genes (Whitfield et al.,
2006). Genotyping arrays can be used to ana-lyze thousands of SNPs in a set of several hun-dred individuals to make a high-density QTL map. SNPs in candidate genes identified by QTL mapping could then be tested for asso-ciation with the trait in populations (Blangero,
2004; Anholt and MacKay,2004).
We believe that MAS will not be a ‘silver bullet’ for making the super-resistant bee. The level of resistance found in A. cerana most surely is regulated by several genes and mark-ers must be developed for several favorable alleles. As genotyping costs continue to fall, MAS may become a useful tool for combining several resistance traits in the same stock.
ACKNOWLEDGEMENTS
Victor Kuznetsov of the Far-Eastern branch of the Russian Academy of Sciences collaborated with all surveys and research in Russia. Nicoloi Kurzenko of the Far-Eastern branch of the Russian Academy of Sciences provided administrative sup-port for work in Russia. Manley Bigalk (IA), Char-lie Harper (LA) and Hubert Tubbs (MS) generously provided test apiaries. We thank one anonymous re-viewer who provided helpful suggestions.
Sélection d’abeilles résistantes à Varroa destruc-tor en Amérique du Nord.
résistance au varroa/ programme de sélection / abeilles russes/ comportement hygiénique sen-sible à varroa/ sélection assistée par marqueur
Zusammenfassung – Zucht auf Resistenz gegen Varroa destructor in Nordamerika. Die Zucht auf Resistenz gegen Varroa destructor in Nordamerika bietet die langfristige Lösung für die von der Mil-be verursachten wirtschaftlichen Schwierigkeiten. Dieses Review untersucht mehrere potenzielle Me-chanismen der Resistenz gegen Varroa und berich-tet über die Entwicklung von zwei Zuchterfolgen, aus denen Bienen von wirtschaftlicher Qualität her-vorgegangen sind, die weniger Pestizidbehandlun-gen gePestizidbehandlun-gen Varroa benötiPestizidbehandlun-gen als unselektierte Bie-nen.
Das VSH Zuchtprogramm konzentriert sich auf die Selektion eines spezifischen Resistenzmecha-nismus, der Varroasensitive Hygiene genannt wird. Das Merkmal VSH wird über den Verkauf von VSH Königinnen, die mit Drohnen bereits vorhandener kommerzieller Linien gepaart wurden, für die Imker verfügbar gemacht. Die größte Resistenz kommt zwar in reinen VSH-Linien vor, die nachhaltigste Verbreitung wird jedoch durch VSH Hybridvölker erzielt. Durch das Auskreuzen reiner VSH Linien mit einer Vielzahl anderer kommerzieller Linien kann die genetische Diversität der Bienenpopula-tion in den USA auf relativ hohem Niveau gehal-ten werden. Reine VSH Zuchtköniginnen werden von Glenn Apiaries produziert und an kommerziel-le Produzenten von Königinnen verkauft, die ihrer-seits ausgekreuzte VSH Königinnen an Imker ver-kaufen.
Putzen, varroasensitive Hygiene und für die Milbe geringe Attraktivität der Brut gehören.
RHB Linien wurden gleichzeitig für Varroaresi-stenz, gute Honigproduktion und Resistenz gegen Tracheenmilben, Acarapis woodi, selektiert. Die Resistenz gegen Tracheenmilben trägt zu ihrer aus-gezeichneten Überwinterungsfähigkeit bei. Der Er-folg der experimentellen RHB Selektion regte eine große kommerzielle Nachfrage an, und RHB wer-den zurzeit von einer als Russian Queen Breeder’s Association bekannten Züchterkooperative gezüch-tet, vermehrt und an die Imker in den USA verbrei-tet.
Die Zucht auf Varroaresistenz wird in der Zu-kunft wahrscheinlich auch markergestützte Selekti-on (MAS) mit einbeziehen, in welcher entweder die Expression von mit Resistenz verbundenen Genen (RNA) oder molekulare Marker, die mit Resistenz-genen in Verbindung stehen (DNA), benutzt werden um die Zuchteltern auszuwählen. Das endgültige Ziel ist, die arbeits- und zeitaufwändige Selekti-on im Feld durch eine Labordiagnose zu ersetzen. Es wird erwartet, dass MAS den Selektionsfort-schritt sowohl für Resistenzmerkmale, die schon entwickelt wurden, als auch für Merkmale, für die diese Entwicklung hin zu nutzbaren kommerziel-len genetischen Linien noch aussteht, wie z.B. ge-genseitiges Putzen und Entfernen von Milben, be-schleunigen wird.
Varroaresistenz / Zuchtprogramm / Russische Honigbienen / Varroasensitive Hygiene / mar-kergestützte Selektion
REFERENCES
Ammons A.D., Hunt G.J. (2008) Identification of quantitative trait loci and candidate genes influ-encing ethanol sensitivity in honey bees, Behav. Genet. 38, 531–553.
Anderson D.L., Trueman J.W.H. (2000) Varroa
jacob-soni (Acari: Varroidae) is more than one species,
Exp. Appl. Acarol. 24, 165–189.
Anholt R.R.H., Mackay T.F.C. (2004) Quantitative genetic analyses of complex behaviours in
Drosophila, Nat. Rev. Genet. 5, 838–849.
Arathi H.S., Ho G., Spivak M. (2006) Inefficient task partitioning among nonhygienic honeybees, Apis
mellifera L., and implications for disease
trans-mission, Anim. Behav. 72, 431–438.
Arechavaleta-Velasco M., Guzman-Novoa E. (2001) Relative effect of four characteristics that restrain the population growth of the mite Varroa
de-structor in honey bee (Apis mellifera) colonies,
Apidologie 32, 157–174.
Arechavaleta-Velasco M.E., Hunt G.J. (2004) Binary trait loci that influence honey bee guarding behav-ior, Ann. Entomol. Soc. Am. 97, 177–183.
Aumeier P. (2001) Bioassay for grooming ef-fectiveness towards Varroa destructor mites in Africanized and Carniolan honey bees, Apidologie 32, 81–90.
Aumeier P., Rosenkranz P. (2001) Scent or movement of Varroa destructor mites does not elicit hygienic behaviour by Africanized and Carniolan honey bees, Apidologie 32, 253–263.
Berg S., Fuchs S., Koeniger N., Rinderer T.E. (2004) Preliminary results on the comparison of Primorski honey bees, Apidologie 35, 552–554. Berg S., Fuchs S., Koeniger N., Rinderer T.E., Büchler
R. (2005) Less mites, less honey–comparing Primorski honey bee lines with Carnica lines in Germany, in: Kaatz H.H., Becher M., Moritz R.F.A. (Eds.), IUSSI Halle, Bees, Ants and Termites –Applied and fundamental research, Regensburg, p. 36.
Bienefeld K., Zautke F., Pronin D., Mazeed A. (1999) Recording the proportion of damaged Varroa
ja-cobsoni Oud. in the debris of honey bee colonies
(Apis mellifera), Apidologie 30, 249–256. Blangero J. (2004) Localization of human
quantita-tive trait loci: King harvest has surely come, Curr. Opin. Genet. Dev. 14, 233–240.
Boecking O., Drescher W. (1992) The removal re-sponse of Apis mellifera L. colonies to brood in wax and plastic cells after artificial and natural infestation with Varroa jacobsoni Oud. and to freeze-killed brood, Exp. Appl. Acarol. 16, 321– 329.
Boecking O., Spivak M. (1999) Behavioral defenses of honey bees against Varroa jacobsoni Oud., Apidologie 30, 141–158.
Bourgeois A.L., Rinderer T.E. (2009) Genetic charac-terization of Russian honey bee stock selected for improved resistance to Varroa destructor, J. Econ. Entomol., in press.
Bourgeois L., Sylvester A., Danka R.G., Rinderer T.E. (2008) Comparison of microsatellite DNA diver-sity among commercial queen breeder stocks of Italian honey bees in the United States and Italy, J. Apic. Res. 47, 93–98.
Brachman B. (2009) Up and running: Russian honey bee breeders, Bee Culture 137, 46–47.
Branco M.R., Kidd N.A.C., Pickard R.S. (1999) Development of Varroa jacobsoni in colonies of
Apis mellifera iberica in a Mediterranean climate,
Apidologie 30, 491–503.
Büchler R. (1990) Possibilities for selecting increased Varroa tolerance in central European honey bees of different origins, Apidologie 21, 365–367. Büchler R. (1997) Field test on Varroa tolerance of the
Kirchhainer population, Apidologie 28, 191–193. Büchler R., Drescher W., Tornier I. (1992) Grooming behaviour of Apis cerana, Apis mellifera and
Varroa jacobsoni and Tropilaelaps clareae, Exp.
Appl. Acarol. 16, 313–319.
Calis J.N.M., Boot W.J., Beetsma J. (2006) Attractiveness of brood cells from different honey bee races (Apis mellifera) to Varroa mites, Proc. Neth. Entomol. Soc. 17, 55–61.
Camazine S. (1986) Differential reproduction of the mite, Varroa jacobsoni (Mesostigmata: Varroidae), on Africanized and European honey bees (Hymenoptera: Apidae), Ann. Entomol. Soc. Am. 79, 801–803.
Chandra S.B.C., Hunt G.J., Cobey S., Smith B.H. (2001) Quantitative trait loci associated with re-versal learning and latent inhibition in honeybees (Apis mellifera), Behav. Genet. 31, 275–285. Crane E. (1978) The Varroa mite, Bee World 59, 164–
167.
Currie R.W., Tahmasbi G.H. (2008) The ability of high- and low-grooming lines of honey bees to re-move the parasitic mite Varroa destructor is af-fected by environmental conditions, Can. J. Zool. 86, 1059–1067.
Danka R., Harris J., Ward K., Ward R. (2008) Status of bees with the trait of Varroa-sensitive hygiene (VSH) for Varroa resistance, Am. Bee J. 148, 51– 54.
Danka R.G., Rinderer T.E., Kuznetsov V.N., Delatte, G.T. (1995) A USDA-ARS project to evaluate re-sistance to V. jacobsoni by honey bees of far-eastern Russia, Am. Bee J. 135, 746–748. Davis A.R. (2009) Regular dorsal dimples on Varroa
destructor – Damage symptoms or developmental
origin? Apidologie 40, 151–162.
de Guzman L.I., Rinderer T.E., Bigalk M., Tubbs H., Bernard S.J. (2005) Russian honey bee (Hymenoptera: Apidae) colonies: Acarapis woodi (Acari: Tarsonemdae) infestations and overwinter-ing survival, J. Econ. Entomol. 98, 1796–1801. de Guzman L.I., Rinderer T.E., Delatte G.T., Stelzer
J.A., Beaman L.D., Harper C. (2002) Hygienic be-havior by honey bees from far-eastern Russia, Am. Bee J. 142, 58–60.
de Guzman L.I., Rinderer T.E., Delatte G.T., Stelzer J.A., Beaman L., Kuznetsov V. (2001) Resistance to Acarapis woodi by honey bees from far-eastern Russia, Apidologie 33, 411–415.
de Guzman L.I., Rinderer T.E., Delatte G.T., Macc-hiavelli R.E. (1996) Varroa jacobsoni Oudemans tolerance in selected stocks of Apis mellifera L., Apidologie 27, 193–210.
de Guzman L.I., Rinderer T.E., Frake A.M. (2007) Growth of Varroa destructor (Acari: Varroidae) populations in Russian honey bee (Hymenoptera: Apidae) colonies, Ann. Entomol. Soc. Am. 100, 187–195.
de Guzman L., Rinderer T., Frake A. (2008) Comp-arative reproduction of Varroa destructor in di ffer-ent types of Russian and Italian honey bee combs, Exp. Appl. Acarol. 44, 227–238.
de Guzman L.I., Rinderer T.E., Lancaster V.A. (1995) A short test evaluating larval attractiveness of honey bees to Varroa jacobsoni Oudemans (Acari: Varroidae), J. Apic. Res. 34, 89–92.
de Guzman L.I., Rinderer T.E., Stelzer J.A. (1997) DNA evidence of the origin of Varroa jacobsoni Oudemans in the Americas, Biochem. Genet. 35, 327–335.
de Guzman L.I., Rinderer T.E., Stelzer J.A. (1999) Occurrence of two genotypes of Varroa jacobsoni Oud. in North America, Apidologie 30, 31–36. Delaplane K.S., Hood W.M. (1999) Economic
thresh-old for Varroa jacobsoni Oud. in the southeastern USA, Apidologie 30, 383–395.
Delaplane K.S., Berry J.A., Skinner J.A., Parkman J.P., Hood W.M. (2005) Integrated pest management against Varroa destructor reduces colony mite lev-els and delays treatment threshold, J. Apic. Res. 44, 157–162.
Donzé G.S., Guerin P.M. (1994) Behavioral attributes and parental care of Varroa mites parasitizing honey bee brood, Behav. Ecol. Sociobiol. 34, 305– 319.
Fries I., Huazen W., Wei S., Jin C.S. (1996) Grooming behavior and damaged mites (Varroa jacobsoni) in
Apis cerana cerana and Apis mellifera ligustica,
Apidologie 27, 3–11.
Fuchs S. (1990) Preference of drone brood cells by
Varroa jacobsoni Oud. in colonies of Apis mellif-era carnica, Apidologie 21, 193–199.
Gary N.E.A. (1960) Trap to quantitatively recover dead and abnormal honey bees from the hive, J. Econ. Entomol. 53, 782–785.
Gary N., Page R.E. (1987) Phenotypic variation in sus-ceptibility of honey bees, Apis mellifera, to in-festation by tracheal mites, Acarapis woodi, Exp. Appl. Acarol. 3, 291–305.
Gilliam M., Taber S. III, Richardson G.V. (1983) Hygienic behavior of honey bees in relation to chalkbrood disease, Apidologie 14, 29–39. Grozinger C.M., Fan Y., Hoover S.E.R., Winston M.L.
(2007) Genome-wide analysis reveals differences in brain gene expression patterns associated with caste and reproductive status in honey bees (Apis
mellifera), Mol. Ecol. 16, 4837–4848.
Harbo J.R. (2001) The relationship between non-reproduction of Varroa and the quantity of worker brood, Am. Bee J. 141, 889–890.
Harbo J.R., Harris J.W. (1999a) Selecting honey bees for resistance to Varroa jacobsoni, Apidologie 30, 183–196.
Harbo J.R., Harris J.W. (2001) Resistance to Varroa
destructor (Mesostigmata: Varroidae) when mite-resistant queen honey bees (Hymenoptera: Apidae) were free-mated with unselected drones, J. Econ. Entomol. 94, 1319–1323.
Harbo J.R., Harris, J.W. (2002) Suppressing mite re-production: SMR an update, Bee Culture 130, 46– 48.
Harbo J.R., Harris J.W. (2005) Suppressed mite repro-duction explained by the behaviour of adult bees, J. Apic. Res. 44, 21–23.
Harbo J.R., Harris, J.W. (2009) Responses to Varroa by honey bees with different levels of Varroa sensitive hygiene, J. Apic. Res. 48, 156–161.
Harbo J.R., Hoopingarner R.A. (1997) Honey bees (Hymenoptera: Apidae) in the United States that express resistance to Varroa jacobsoni (Mesostigmata: Varroidae), J. Econ. Entomol. 90, 893–898.
Harris J.W. (2007) Bees with Varroa sensitive hygiene preferentially remove mite infested pupae aged five days post capping. J. Apic. Res. 46, 134–139. Harris J.W. (2008) Effect of brood type on Varroa-sensitive hygiene by worker honey bees (Hymenoptera: Apidae), Ann. Entomol. Soc. Am. 101, 1137–1144.
Harris J.W., Harbo, J.R. (1999) Low sperm counts and reduced fecundity of mites in colonies of honey bees (Hymenoptera: Apidae) resistant to
Varroa jacobsoni (Mesostigmata: Varroidae), J.
Econ. Entomol. 92, 83–90.
Harris J.W., Harbo J.R. (2000) Changes in reproduc-tion of Varroa destructor after honey bee queens were exchanged between resistant and susceptible colonies, Apidologie 31, 689–699.
Harris J., Rinderer T.E. (2004) Varroa resistance in hy-brid ARS Russian honey bees, Am. Bee J. 144, 797–799.
Harris J.W., Danka R.G., Villa J.D. (2009) Hygienic activity toward Varroa mites in capped brood is not dependent on mite reproductive status, Am. Bee J. 149, 587–588.
Harris J.W., Danka R.G., Villa J.D. (2010) Honey bees (Hymenoptera: Apidae) with the trait of Varroa Sensitive Hygiene remove brood with all repro-ductive stages of Varroa mites (Mesostigmata: Varroidae), Ann. Entomol. Soc. Am. 103, 146– 152.
Harris J.W., Harbo J.R., Villa J.D., Danka R.G. (2003) Variable population growth of Varroa destructor (Mesotigmata: Varroidae) in colonies of honey bees (Hymenoptera: Apidae) during a 10-year pe-riod, Environ. Entomol. 32, 1305–1312.
Harris J., Rinderer T.E., Kuzentsov K., Danka G., De Guzman L.I., Villa J. (2002) Imported Russian honeybees: Quarantine and initial selection for Varroa resistance, Am. Bee J. 142, 591–596.
Haydak M.H. (1945) The language of the honey bees, Am. Bee J. 85, 316–317.
Honey Bee Genome Sequencing Consortium (2006) Insights into social insects from the genome of the honey bee, Apis mellifera, Nature 443, 931–949. Hospital F. (2009) Challenges for effective
marker-assisted selection in plants, Genetica 136, 303– 310.
Hunt G.J., Page R.E. Jr. (1995) A linkage map of the honey bee, Apis mellifera, based on RAPD mark-ers, Genetics 139, 1371–1382.
Hunt G.J., Amdam G.V., Schlipalius D., Emore C., Sardesai N., Williams C.E., Rueppell O., Guzmán-Novoa E., Arechavaleta-Velasco M., Chandra S., Fondrk M.K., Beye M., Page R.E. Jr. (2007) Behavioral genomics of honeybee foraging and nest defense, Naturwissenschaften 94, 247–267. Hunt G.J., Guzmán-Novoa E., Fondrk M.K., Page R.E.
Jr. (1998) Quantitative trait loci for honey bee stinging behavior and body size, Genetics 148, 1203–1213.
Hunt G.J., Page R.E. Jr., Fondrk M.K., Dullum C.J. (1995) Major quantitative trait loci affecting honey bee foraging behavior, Genetics 141, 1537–1545. Ibrahim A., Spivak M. (2006) The relationship
be-tween hygienic behavior and suppression of mite reproduction as honey bee (Apis mellifera) mechanisms of resistance to Varroa destructor, Apidologie 37, 31–40.
Ibrahim A., Reuter G.S., Spivak M. (2007) Field trial of honey bee colonies bred for mechanisms of re-sistance against Varroa destructor, Apidologie 38, 67–76.
Kralj J. (2004) Parasite-host interactions between
Varroa destructor Anderson and Trueman and Apis mellifera L.: Influence of parasitism on flight
behaviour and the loss of infested foragers, Johann Wolfgang Goethe University of Frankfurt am Main, PhD Dissertation.
Kralj J., Fuchs S. (2006) Parasitic Varroa destructor mites influence flight duration and homing ability of infested Apis mellifera foragers, Apidologie 37, 577–587.
Lapidge K.L, Oldroyd B.P., Spivak M. (2002) Seven suggestive quantitative trait loci in-fluence hygienic behavior of honey bees, Naturwissenschaften 89, 565–568.
Lodesani M., Vecchi M.A., Tommasini S., Bigliardi M. (1996) A study on different kinds of dam-age to Varroa jacobsoni in Apis mellifera ligustica colonies, J. Apic. Res. 35, 49–56.
McFarlane J.E. (1976) The influence of dietary cop-per and zinc on the growth and reproduction of the house cricket, Can. Entomol. 108, 387–390. Mondragón L., Spivak M., Vandame R. (2005) A
mul-tifactorial study of the resistance of honeybees
Apis mellifera to the mite Varroa destructor over
one year in Mexico, Apidologie 36, 345–358. Moretto G., Gonçalves L.S., de Jong D. (1993)
Heritability of Africanized and European honey bee defense behavior against the mite Varroa
ja-cobsoni, Braz. G. Genet. 16, 71–77.
Morse R.A., Miksa D., Masenheimer J.A. (1991) Varroa resistance in the US honey bees, Am. Bee J. 131, 433–434.
Navajas M., Migeon A., Alaux C., Martin-Magniette M.L., Robinson G.E., Evans J.D., Cros-Arteil S., Crauser D., Le Conte Y. (2008) Differential gene expression of the honey bee Apis mellifera as-sociated with Varroa destructor infection, BMC Genomics 9, 301.
Nazzi F., Milani N., Vedova G.D., Nimis, M. (2001) Semiochemicals from larval food affect the locomotory behaviour of Varroa destructor, Apidologie 32, 149–155.
Oxley P.R., Thompson G.J., Oldroyd B.P. (2008) Four quantitative trait loci that influence worker steril-ity in the honeybee (Apis mellifera), Genetics 179, 1337–1343.
Page R.E. Jr., Fondrk M.K., Hunt G.J., Guzmán-Novoa E., Humphries M.A., Nguyen K., Greene A.S. (2000) Genetic dissection of honeybee (Apis
mel-lifera L.) foraging behavior, J. Hered. 91, 474–
479.
Peng Y.S., Fang, Y., Xu S., Ge L., Nasr M.E. (1987) Response of foster Asian honeybee (Apis cerana Fabr.) colonies to the brood of European honey-bee (Apis mellifera L.) infested with parasitic mite,
Varroa jacobsoni Oudemans, J. Invertebr. Pathol.
49, 259–264.
Rinderer T.E., Delatte G.T., de Guzman L.I., Williams J., Stelzer J.A., Kuznetsov, V. (1999) Evaluations of the Varroa-resistance of honey bees imported from far-eastern Russia, Am. Bee J. 139, 287–290. Rinderer T.E., de Guzman L.I., Delatte G.T., Stelzer J.A., Lancaster V.A., Kuznetsov V., Beaman L., Watts R., Harris, J.W. (2001a) Resistance to the parasitic mite Varroa destructor in honey bees from far-eastern Russia, Apidologie 32, 381–394. Rinderer T.E., de Guzman L.I., Delatte G.T., Stelzer J.A., Williams J.L., Beaman L.D., Kuznetsov V., Bigalk M., Bernard S.J., Tubbs H. (2001b) Multi-state field trials of Russian honey bees 1. Responses to Varroa destructor 1999, 2000, Am. Bee J. 141, 658–661.
Rinderer T.E., de Guzman L.I., Delatte G.T., Stelzer J.A., Lancaster V.A., Williams J.L., Beaman, L.D., Kuznetsov V., Bigalk M., Bernard S.J., Tubbs H. (2001c) Multi-State Field Trials of Russian Honey
Bees: 2. Honey Production 1999, 2001, Am. Bee J. 141, 726–729.
Rinderer T.E., de Guzman L.I., Delatte G.T., Harper C. (2003) An evaluation of ARS Russian honey bees in combination with other methods for the control of Varroa mites, Am. Bee J. 143, 410–413. Rinderer T.E., de Guzman L., Harper C. (2004) The
effects of co-mingled Russian and Italian honey bee stocks and sunny or shaded apiaries on Varroa mite infestation level, worker bee population and honey production, Am. Bee J. 144, 481–485. Rosenkranz P., Fries I., Boecking O., Sturner M.
(1997) Damaged Varroa mites in the debris of honey bee (Apis mellifera L.) colonies with and without hatching brood, Apidologie 28, 427–437. Rothenbuhler W.C. (1960) A technique for studying genetics of colony behavior in honey bees, Am. Bee J. 100, 176,198.
Rothenbuhler W.C. (1964) Behavior genetics of nest cleaning in honey bees: IV. Responses of F1
and backcross generations to disease-killed brood, Am. Zool. 4, 111–123.
Rüeppell O., Chandra S., Pankiw T., Fondrk M.K., Beye M., Hunt G.J., Page R.E. Jr. (2006) The ge-netic architecture of sucrose responsiveness in the honey bee (Apis mellifera L.), Genetics 172, 243– 251.
Rüeppell O., Pankiw T., Nielsen D.I., Fondrk M.K., Beye M., Page R.E. (2004) The genetic architec-ture of the behavioral ontogeny of foraging in hon-eybee workers, Genetics 167, 1767–1779. Ruttner F., Hänel H. (1992) Active defense against
Varroa mites in a Carniolan strain of honeybee
(Apis mellifera carnica Pollman), Apidologie 23, 173–187.
Solignac M., Vautrin D., Baudry E., Mougel F., Loiseau A., Cornuet J.-M. (2004) A microsatellite-based linkage map of the hon-eybee, Apis mellifera L., Genetics 167, 253–262. Spivak M. (1996) Honey bee hygienic behavior and
defense against Varroa jacobsoni, Apidologie 27, 245–260.
Spivak M., Reuter G.S. (1998) Honey bee hygienic be-havior, Am. Bee J. 138, 283–286.
Spivak M., Reuter G.S. (2001a) Varroa jacobsoni in-festation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior, J. Econ. Entomol. 94, 326–331.
Spivak M., Reuter G.S. (2001b) Resistance to American foulbrood disease by honey bee colonies, Apis mellifera, bred for hygienic behav-ior, Apidologie 32, 555–565.
Szabo T I., Walker C.R.T. (1995) Damages to dead
Varroa jacobsoni caused by the larvae of Galleria mellonella, Am. Bee J. 135, 421–422.
Thakur R.K., Bienefeld K., Keller R. (1997) Varroa de-fense behavior in A. mellifera carnica, Am. Bee J. 137, 143–148.
Tubbs H., Harper C., Bigalk M., Bernard S.J., Delatte G.T., Sylvester H.A., Rinderer T.E. (2003) Commercial management of ARS Russian honey bees, Am. Bee J. 144, 819–820.
Vandame R., Morand S., Colin M.-E., Belzunces L.P. (2002) Parasitism in the social bee Apis mellifera: quantifying costs and benefits of behavioral resis-tance to Varroa destructor mites, Apidologie 33, 433–445.
Villa J.D. (2006) Autogrooming and bee age influence migration of tracheal mites to Russian and sus-ceptible worker honey bees (Apis mellifera L.), J. Apic. Res. 45, 28–31.
Villa J.D., Rinderer T.E. (2008) Inheritance of resis-tance to Acarapis woodi (Acari: Tarsonemidae) in crosses between selected resistant Russian and se-lected susceptible US honey bees (Hymenoptera: Apidae), J. Econ. Entomol. 101, 1756–1759. Villa J.D., Danka R.G., Harris J.W. (2009a) Simplified
methods of evaluating colonies for levels of Varroa sensitive hygiene (VSH), J. Apic. Res. 48, 162– 167.
Villa J.D., Rinderer T.E., Bigalk M. (2009b) Overwintering of Russian honey bees in north-eastern Iowa, Science of Bee Culture 1, 19–21, Suppl. to Bee Culture 137.
Ward K., Danka R., Ward R. (2008) Comparative per-formance of two mite-resistant stocks of honey bees (Hymenoptera: Apidae) in Alabama beekeep-ing operations, J. Econ. Entomol. 101, 654–659. Webster T.C., Thacker E.M., Vorisek F.E. (2000)
Live Varroa jacobsoni (Mesostigmata: Varroidae) fallen from honey bee (Hymenoptera: Apidae) colonies, J. Econ. Entomol. 93, 1596–1601. Whitfield C.W., Behura S.K., Berlocher S.H., Clark
A.G., Johnston J.S., Sheppard W.S., Smith D.R., Suarez A.V., Weaver D., Tsutsui N.D. (2006) Thrice out of Africa: Ancient and recent expan-sions of the honey bee, Apis mellifera, Science 314, 642–645.
Whitfield C.W., Cziko A.M., Robinson G.E. (2003) Gene expression profiles in the brain predict be-havior in individual honey bees, Science 302, 296– 299.
